Breast cancer is caused by the accumulation of genetic and epigenetic changes. The cDNA microarray technology has been widely used for identifying cancer-related changes in gene expression for breast tumor classification and prognosis. However, the information obtained from cDNA microarray analysis is not sufficient to accurately characterize the biological state of gene products. The profiling of cancer-related changes in protein functions is much informative in elucidating the molecular mechanism by which cancer occurs. Transcription factors are important cellular proteins, which regulate gene expression. We have recently developed protein/DNA array technology that is capable of profiling the activation of multiple transcription factors simultaneously. We propose to verify, in the phase I study, if this novel array technology can be used to identify cancer-related patterns of TF activity by profiling the activities of 300 transcription factors in breast cancer cell lines. We will develop (1) a glass microarray to replace the current membrane-based array and (2) nitrocellulose-based spin column to replace agarose gel separation of protein/DNA complexes. With the improvements, the entire assay procedures can be automated, which will make it much easier to analyze a lot of samples simultaneously. We will apply this technology to analyze 20 breast cancer cell lines that are derived from different stages of breast cancers as well as one normal breast cell line and to compare the patterns of transcription factor activity among them in order to identify the signature of transcription factors for breast cancers.